Saturable and stereospecific binding sites for tritium labeled threo-(plus or minus)-methylphenidate were characterized in rat brain membranes. The highest density of labeled threo-(plus or minus)-methylphenidate binding sites was found in the synaptosomal fraction of corpus striatum. Scatchard analysis revealed a single class of non-interacting binding sites with an apparent dissociation constant (Kd) of 235 nM and a maximum number of binding sites (Bmax) of 13.4 pmol/mg protein. Saturable, high affinity binding of labeled threo-(plus or minus)-methylphenidate to striatal synaptosomes was dependent upon the presence of sodium ions. A good correlation (r = 0.88; p less than 0.001) was observed between the potencies of various psychotropic drugs in displacing labeled threo-(plus or minus)-methylphenidate from these sites and their potencies as inhibitors of (H3)dopamine uptake into striatal synaptosomes. A good correlation (r = 0.85; p less than 0.001) was also observed between the potencies of a series of ritalinic acid esters in inhibiting labeled threo(plus or minus)-methylphenidate binding to striatal synaptosomes and their potencies as motor stimulants in mice. These observations suggest that the binding sites for labeled threo-(plus or minus)-methylphenidate described here are associated with a dopamine uptake or transport complex, and that these sites may mediate the motor stimulant properties of ritalinic acid esters such as methylphenidate.